1. Field of the Invention
The present invention, primer generation-rolling circle amplification (PG-RCA), allows the single step detection of nucleic acid sequences such as DNA and RNA sensitively and rapidly. Furthermore, this technology is easily applicable to detection of other biomolecules, such as DNA methylation, single nucleotide polymorphisms (SNP), proteins and posttranslational modifications. The invention also relates to a ribbon probe useful in PG-RCA.
2. Description of the Related Art
A variety of nucleic acid sequence detection technologies have been established in recent years for in vitro diagnostics. Several of these technologies are PCR (polymerase chain reaction), LCR (Ligase chain reaction), RCA (rolling circle amplification), NASBA (nucleic acid sequence based amplification) and TMA (transcription mediated amplification). Some of these technologies allow detection of even less than 10 molecules through their exponential amplification mechanism.
In pathogen detection, these amplification technologies are widely utilized to detect contamination by pathogens in a sample by targeting pathogen-specific sequences or genes found in a pathogen's genomic DNA. However, as a single cell contains only a single set of genomic DNA, these nucleic acid amplification-based pathogen assays have only comparable sensitivity to other conventional assays such as enzyme linked immunosorbent assay (ELISA). One solution is to target more abundant pathogen-specific rRNA or mRNA, but many of the nucleic acid amplification reactions utilize DNA polymerase, which cannot polymerize on RNA templates efficiently, therefore RNA targets cannot be directly detected in many of these amplification methods.
Reverse transcription-polymerase chain reaction (RT-PCR) is most widely used to detect target RNA sequence through conversion of RNA sequences to single strand or double strand DNA by reverse transcriptase followed by PCR amplification. However, as RT and PCR need to be conducted separately due to the difference in their reaction conditions, RT-PCR takes a few hours in total to obtain a detectable amount of product and sometimes requires transferring RT product into PCR, which is very tedious when there are many samples.
Ligation-rolling circle amplification is another way to detect target RNA sequence through ligation of the 5′ and 3′ ends of a padlock probe or linear nucleic acid probe on the target RNA followed by hyperbranched rolling circle amplification (HRCA) to quantitate the circularized padlock probe. However, this method requires at least two reactions, ligation and HRCA, and may require exonuclease treatment to remove uncircularized padlock probes after circularization for an efficient HRCA reaction. Furthermore, DNA ligase-based ligation on an RNA template is known to be less efficient than on a DNA template, therefore small amounts of target RNA may not be detected.